Stractural and molecular biological study of the ciliary basal apparatus during ciliogenesis

1.我们获得了6株标记与纤毛伸展的基底体相关的纹状根的单抗。这些抗体通过免疫印迹鉴定出205-215 kDa的蛋白。利用克隆R67，我们揭示了与孤立纤毛相关的条纹细根的复杂形态。205-215抗原是小根暗条纹的组成成分。在人颌下腺的所有细胞类型中，纹状根均呈阳性染色。结果表明：1.纹状根与中心粒的定位有关，在正常生理条件下可能与中心粒有关，不形成孤立纤毛。秋水仙胺处理后，PTK_2细胞内中心粒相关的小根伸长和/或结构复杂化，胞质内游离小根组织起来。这些变化是可逆的，停药2小时后几乎消失。这些观察结果可能表明了细胞质微管和有条纹的小根之间的功能关系，以及细胞质中除中心粒外还存在着小根成核因子。用荧光免疫组织化学和免疫电子显微镜技术研究了γ-微管蛋白在睫状体基底器中的定位。除基底体外，外侧基底足尤其是基底帽中还发现了γ-微管蛋白，证实了先前的观察结果，即基底足是微管的组织中心。在延伸的单纤毛细胞中，基体和单个中心粒由一束或两束细丝相连，并有少量的周期性条纹。我们将这些捆绑称为条纹连接器。该连接体对稳定顶端细胞质中的基底体有重要作用。

1. We obtained six monoclonal antibodies that labeled striated rootlets associated with basal bodies extending cilia. These antibodies identified proteins of 205-215 kDa by immunoblotting. Using a clone R67, we revealed the complicated morphology of striated rootlets associated with solitary cilia. The 205-215 antigens were constitutive components of dark striations of the rootlet. Striated rootlets were positively stained in all cell types in the human submandibular glands. It is suggested that striated rootlets are concerned with the positioning of centrioles and may be associated with centrioles under normal physiological conditions, without formation of solitary cilia.2. Treatment with colcemid resulted in the elongation and/or structural complication of the centriole-associated rootlets in PtK_2 cells and additionally, organization of intracytoplasmic free rootlets. The changes were reversible and almost disappeared 2 hours after removal of the drug. These observations may indicate functional relationships between cytoplasmic microtubules and striated rootlets, and also the existence of rootlet-nucleating factors in the cytoplasm in addition to centrioles.3. The localization of gamma-tubulin in the ciliary basal apparatus was studied by fluorescence immunohistochemistry and immunoelectron microscopy. Ip addition to basal bodies, gamma-tubulin was identified in the lateral basal foot, especially the basal foot cap, confirming the previous observations that the basal foot serves as the microtubule organizing center.4. In the cells extending solitary cilia, the basal body and the single centriole were interconnected by one or two bundles of thin filaments with a few periodic striations. We have called these bundles the striated connector. The connector could play an important role for stabilising the basal body in the apical cytoplasm.